Helicobacter pylori adhesin binding group antigen

ABSTRACT

A novel blood group antigen binding (BAB) adhesin protein was isolated and purified, whereby said protein or fractions thereof bind specifically to  Helicobacter pylori  fucosylated blood group antibodies. Said adhesin has a molecular weight of about 73.5 kDa and the N-terminal sequence for the adhesin and the corresponding DNA show homologies between different strains of  H. pylori.  Said adhesin and/or DNA is useful for diagnose and therapy and/or profylax directed against  H. pylori  induced infections, e.g. gastritis and acid peptic disease.

This application is the national phase under 35 U.S.C. §371 of prior PCTInternational Application No. PCT/SE97/01009 which has an Internationalfiling date of Jun. 10, 1997 which designated the United States ofAmerica.

FIELD OF THE INVENTION

The present invention relates to materials and methods for prevention,treatment and diagnosing of infections caused by Helicobacter pylori.More specifically the present invention relates to polypeptides andantibodies useful in vaccines for the treatment and prevention ofpathologic infections caused by Helicobacter pylori strains. The presentinvention specifically relates to a bacterial blood group antigenbinding adhesin (BAB-adhesin). The present invention further relates topolynucleotides useful for the recombinant production of saidpolypeptides and for use in immunisation therapies. In addition, itrelates to polypeptides, antibodies, and polynucleotides used for thedetection of said bacteria.

The present invention further relates to new immunoglobulins, whichexhibit specific activity to a blood group binding adhesin, expressed byHelicobacter pylori, methods for the production of said immunoglobulins,their isolation and use. The present invention further relates to thetreatment and prevention of H. pylori induced infections in thegastrointestinal tract.

BACKGROUND OF THE INVENTION

Helicobacter pylori is a causative agent for acid peptic disease and thepresence of this organism is highly correlated to the development ofgastric adenocarcinoma. Bacterial adherence to the human gastricepithelial lining was recently shown to be mediated by fucosylated bloodgroup antigens.

Recent research has focused on the role of Helicobacter pylori in thedevelopment of ulcers in the gastric mucosa. Recent findings show astrong connection between H. pylori and chronic, active gastritis andgastric ulcers. Furthermore, there appears to be a strong correlationbetween ventricular cancer and gastric ulcers. Traditional treatment ofgastric ulcers has involved gastric resection, the administration ofbismuth compositions, the administration of H₂-blockers and theadministration of pH-buffering agents, to mention a few examples.

More recently, various forms of treatment have been supplemented withthe administration of antibiotics. One problem with presently knowntreatments is the risk for treatment failure. Furthermore, not only domicrobes develop antibiotic resistance, the antibiotics administeredoften upset the natural balance of benign microbes, colonising theintestinal tract. This leads to diarrhoea and other signs of intestinaldiscomfort, in addition to destabilising the benign flora in theintestines. Other treatments, e.g. H₂-blockers often require life-longmedication to prevent the recurrence of disease.

The foregoing, together with the fact that H. pylori is very widelyspread among humans—according to a conservative estimate approximately60% of all adult humans in the industrialised countries areinfected—makes the diagnosing, prevention and treatment of H. pyloriinfections an urgent task.

Further, the fact that developing countries frequently lack theresources for conventional treatment of gastric ulcers furtherunderlines the importance of finding new ways of treatment andprevention of H. pylori induced infections. It is obvious, for manyreasons, that disease prevention with vaccines is a preferable mode. Avaccine would provide an easily administered and economical prophylacticregimen against H. pylori infections. An effective vaccine against H.pylori is nevertheless presently lacking.

STATE OF THE ART

H. pylori colonises the human gastric mucosa, in an equilibrium betweenadherence to the epithelial surface mucous cells and the mucous layerlining the gastric epithelium. Once infected, bacteria seems to colonisefor a lifetime. Attachment to the epithelial lining protects thebacteria from the anti-microbial effects of the acidic gastric juice ofthe stomach lumen, as well as from physical forces such as peristalsis.For survival in this hostile ecological niche, H. pylori has developed abattery of virulence factors; such as production of the enzyme ureasethat buffers the micro-environment around the bacteria and the polarflagellae to ensure high motility, a prerequisite in an ecological nichewhere the turnover of the mucous layer is in the range of hours. Asubset of H. pylori strains produces the vacuolating cytotoxin, VacA,and the cytotoxin associated antigen CagA.

Attachment is essential for colonisation of the epithelial lining andbacteria express surface associated adhesion molecules that recognisespecific carbohydrate or protein receptors on the cell surfaces ormucous lining. The specificity in this interaction in combination withthe genetically regulated receptor distribution results in a restrictedrange of cell lineages and tissues available for colonisation. Severalputative receptor structures have been described for H. pylori, such asthe hemagglutinin-sialic acid, sulphated glycoconjugates andsulphatides. Recently, the fucosylated blood group antigens H-1 andLewis^(b) were described (Borén et al., Science, 262, 18921993),mediating specific adherence of H. pylori to human and rhesus monkeygastric surface mucous cells in situ. The H-1 and Lewis^(b) antigens arepart of the blood group antigens that define blood group O in the ABOsystem.

Surface-exposed proteins are often constituents of the outer membrane.The outer membrane has a structural role and acts as a selectivebarrier, determining what enters the cell and what molecules aresecreted. One class of outer membrane proteins are called porins, andcreate hydrophilic pores through the outer membrane where specificmetabolites, such as sugar molecules, can cross. Recently the finding ofa number of outer membrane proteins in H. pylori, was reported, whichproteins were suggested to constitute a family of porin proteins.

The BAB adhesin has previously been identified and shown to be localisedon the bacterial surface of H. pylori (SE 9602287-6). The blood groupbinding activity was shown to be pH dependent and the present inventorspresent evidence that the binding affinity to the Lewis^(b) receptorreveals a high equilibrium constant. For the purification of the BABadhesin, a crosslinker-labelled receptor conjugate was used in order tomediate specific transfer of biotin to the adhesins on the bacterialsurface. Thereafter the biotin-labelled adhesin could be extracted bystreptavidin coated magnetic beads. Determination of the amino terminalamino acid sequence of the purified BAB adhesin exhibit homologies toouter membrane proteins of H. pylori porins.

Intensive research has been directed to the immunological treatment andprevention of H. pylori induced infections. EP 0 484 148 (Ando &Nakamura) describes a method for treating and/or preventing uppergastrointestinal disease in mammals, said method comprising orallyadministering to a patient in need thereof an effective amount of apharmaceutical composition comprising anti-Helicobacter pyloripolyclonal immunoglobulins and a pharmaceutically acceptable carrier.Said description further dwells on the combination of said treatment incombination with the administration of antibiotics. As the method ofproducing said polyclonal antibodies, EP 0 484 148 describes theisolation and purification of anti-H. pylori immunoglobulins from thesera and milk of mammals. H. pylori itself was not found in the stomachsof cows, goats, sheep, swine or horses, according to EP 0 484 148, butit was assumed that these animal species have colonizing microorganismswith antigenic determinants similar to those of H. pylori because theyhave immunoglobulins which cross-react to strains of H. pylori found inhumans. Preferably, according to EP 0 484 148, large mammals, e.g.pregnant cows, are immunized with whole cells of H. pylori and theimmunoglobulins subsequently extracted from the milk or colostrum. Inthe immunization experiments, NCTC Strain 11362 and clinical isolate H.pylori No. 153 were used to trigger the production of immunoglobulins.On the other hand, NCTC Strain 11637 was used for analysing purposes.Immunization is claimed to yield an anti-H. pylori titer in the milk ofsuch magnitude, that daily doses of 0.01-0.1 g/day immunoglobulincomposition, are sufficient for successful therapy. The claimed intervalof 0.01-0.1 g/day is however not supported by the experiments presentedby Ando & Nakamura and so low doses have hitherto not proven efficientin clinical tests. The doses actually used in example 5 and 7 are in theorder of magnitude of 1 g/day, i.e. 10-fold the upper limit of the giveninterval. Furthermore, it is very unlikely, that unspecificimmunoglobulin mixtures as those manufactured by Ando & Nakamura, wouldbe effective in claimed doses as similar doses are ineffective againstother gastrointestinal pathogens. The simultaneous administration ofantibiotics, extensively discussed in the description, underlines theinsufficiency of the disclosed immunoglobulins.

EP 0 469 359 (Cordle & Schaller) likewise describes the immunization ofmammals, preferably pregnant cows, with formalin killed H. pyloribacteria (ATCC Strain 26695). Anti-H. pylori polyclonal antibodies wereisolated and purified from the milk and finally fed to piglets, inamounts of about 0.5 g immunoglobulins, three times daily. The resultswere assessed by determination of the number of biopsy specimens, whichwere positive for Gram-negative bacteria after the trial. Gram-negativebacteria was found in 78% of the piglets fed a non-immune nutrient butonly (Sic!) in 35% of the piglets fed a nutrient containing so calledspecific anti-H. pylori antibodies.

Anti-H. pylori polyclonal antibodies, effective to cause aggregation ofH. pylori, have thus been administered orally as a regimen in thetreatment and prevention of H. pylori induced infections in thegastrointestinal tract. Nevertheless, as also noted in EP 0 484 148 A1,it is still not clear, how many antigenic determinants are present onthe surface of H. pylori. The occurrence of a wide variety of H. pyloristrains, makes questionable the practical efficiency of any polyclonalimmunological therapy according to the state of the art. Immunizationusing whole bacteria will always trigger a highly polyclonalimmunresponse with a low level of antibodies against a given antigenicdeterminant. This is underlined e.g. by the results presented by Cordle& Schaller, where, although the number of Helicobacter positive biopsieswere reduced, complete cure was not obtained through the treatmentaccording to their invention.

It is notable, that the dose of immunoglobulin needed for oralprophylaxis or therapy has not yet been clearly defined. In a normalhuman adult, approximately 5 g IgA is produced and secreted at mucosalsurfaces each day. Obviously, doses of this magnitude are economicallyand practically unfeasible for large-scale therapy or prophylaxis. Instudies on the effect of oral immunoglobulin on rotavirus infection,daily doses in the interval of 600 to 9000 mg have been tried inclinical tests. Successful intervention has also been reported whentreating H. pylori and cryptosporidial infections with dailyadministrations of 3 to 15 g immunoglobulin from immunized cows(Hammarström et al., Immunol Rev, 139 (1994) 43-70). Generally speaking,all studies hitherto point to the necessity of using high doses ofimmunoglobulins when trying to combat an ongoing infection. The need formore specific immunoglobuline preparations, allowing the use of smallerdoses, is thus an urgent one.

To maximize the potency of an immunological regimen for the treatmentand prevention of H. pylori, it is of great importance to find aspecific conserved antigenic determinant, which plays a central role forthe pathogenicity of H. pylori. Using such an antigenic determinantwould make it possible to produce highly specific and therapeuticallyefficient novel polyclonal and/or monoclonal immunoglobulinpreparations.

SUMMARY OF THE INVENTION

The above problem of providing specific, cost-efficient andtherapeutically superior immunoglobulin preparations for the treatmentand prevention of H. pylori has now been solved through the compositionand methods according to the attached patent claims. The presentinventors have now surprisingly shown, that highly specific andtherapeutically efficient polyclonal and/or monoclonal immunoglobulinpreparations can be provided through the immunization of an animal withan adhesin protein, specific for H. pylori. The invention will now bedescribed in closer detail with reference to the attached, non-limitingfigures and examples.

One objective of the present invention was to further purify andcharacterize the H. pylori blood group antigen binding (BAB) adhesin tomake possible the development of methods and materials for specific andselective diagnosing and treatment of H. pylori induced infections andrelated diseases and the development of said methods and materials. Afurther and equally important objective was to determine the DNAsequences of the genes involved in the expression of this protein. Theseobjectives were fulfilled through the protein, the DNA and the methodsand materials specified herein. The DNA sequences are attached as SEQ IDNOS: 1 and 2, disclosing the babA and babB sequences, respectively. Thefull protein sequences are disclosed in SEQ ID NOS: 3 and 4.

DESCRIPTION OF THE FIGURES

FIG. 1A) illustrates the bacterial binding to soluble blood groupantigens. H. pylori strains were incubated with ¹²⁵I-labeled blood groupantigen glycoconjugates and bound ¹²⁵I-activity was measured (Note theabsence of blood group antigen binding shown for strains MO19 and26695),

FIG. 1B) illustrates an receptor displacement assay. Strain CCUG 17875was first incubated with 10 ng ¹²⁵I-labeled Le^(b) antigenglycoconjugate and the complex was then challenged (1 h) with an excessof unlabeled Le^(b) or Le^(a) glycoconjugate, before the ¹²⁵I-activityin the bacterial pellet was measured. Concentrations of the unlabeledglycoconjugate ranged from 50 ng to 8 μg and

FIG. 1C) shows the results of a Scatchard analysis of the H.pylori-Le^(b) antigen interaction. Bacterial binding to the Le^(b)glycoconjugate was titrated to an affinity constant (Ka) value of8×10⁻¹⁰ M⁻¹ (13).

FIGS. 2A-D: Upper panel: Prevalence of the BabA adhesin in the bacterialpopulation. Cells of strain CCUG 17875 were incubated with biotinylatedLe^(b) FIG. 2(A) or Le^(b) FIG. 2(B) glycoconjugate. Bound biotinylatedLewis-conjugate was detected with FITC-labeled streptavidin (greenfluorescence) and bacteria were counterstained with propidium iodine(red fluorescence). Lower panel: Localisation of the BabA adhesin. Forelectron microscopy (15) cells of strain CCUG 17875 were incubated withbiotinylated Le^(b) FIG. 2(C) or Le^(a) FIG. 2(D).

FIGS. 3A-C shows the characterization of the molecular weight of theBabA adhesin by the use of receptor overlay analysis FIGS. 3(A, B) andreceptor activity directed affinity tagging of BabA FIG. 3(C).

FIGS. 4A-C shows receptor activity directed affinity tagging and proteinpurification of the BabA adhesin.

FIG. 5 shows the translated amino acid sequences for the babA and babBgenes, corresponding to the N-terminal domain of the BabA (SEQ ID NOS: 7and 8) adhesin.

FIG. 6 shows the procentual inhibition of H. pylori binding to¹²⁵I-labeled Lewis b antigen for different preparations as a function ofthe antibody titre.

FIG. 7 shows a Western blot detection of the BabA adhesin by thedifferent antibody preparations.

FIG. 8 shows four Western blot analyses of H. pylori proteins by thedifferent antibody preparations.

DESCRIPTION OF THE INVENTION

The blood group antigen binding adhesin, BabA, has now beenbiochemically characterized and purified by a novel technique, receptorActivity Directed Affinity Tagging (Retagging). Two genes, babA and babBwere found to code for two different but very similar proteins. Thepresent invention thus comprises a novel blood group antigen bindingadhesin. The DNA sequences are disclosed in SEQ ID NO: 1 (babA) and SEQID NO: 2 (babB) The protein sequences are disclosed in SEQ ID NOS: 3 and4. The invention also includes any pharmaceutical composition comprisingsaid adhesin protein and/or fractions thereof. Examples of suchpharmaceutical compositions are for example medicaments for theprevention or treatment of Helicobacter pylori induced gastritis,gastric and duodenal ulcers and gastric adenocarcinoma. Optionally saidpharmaceutical composition additionally encompasses pharmaceuticallyacceptable excipients.

Further, the present invention comprises the BAB-adhesin gene or genesfor expression of an adhesin protein according to the invention. Saidinvention also comprises a novel method for the isolation andpurification of said adhesin. The disclosed genes are contemplated tofunction as a cassette system, the organism alternating between these toavoid immunity in the host. It is very likely, that homologies of thedisclosed sequences exist and additionally supplement said cassettefunction in other strains of H. pylori. Also, genes corresponding to ahomology of the first 40 amino acids or genes, corresponding to ahomology of the last, about 300 amino acids, can function to thiseffect. It is further highly likely, that Helicobacter pylori is able toswitch between several genes, similar to the disclosed genes, in aso-called cassette system.

The invention additionally comprises monospecific antisera producedusing the novel adhesin protein and/or fractions thereof. Saidmonospecific antisera is preferably produced according to any suitable,conventional method for producing monospecific antisera in vitro or invivo, e.g. by inoculating a suitable animal. Such methods are familiarto a person skilled in the art. Antibodies raised in a suitable animalor in the patient to be treated, can subsequently be administeredlocally, e.g. orally to the patient.

The invention further comprises the use of said monospecific antiserafor the manufacturing of a test kit for quantitative or qualitativedeterminations of adhesin protein or fractions thereof in cells, tissuesor body fluids.

The invention further comprises the use of said adhesin protein orcorresponding DNA for use in therapy or immunisation and/or in themanufacture of compositions for said uses. The invention specificallyencompasses the use of said DNA for immunisation therapy and for themanufacture for compositions for such therapy. Preferably, in animmunisation therapy where said composition is administered orally to apatient, the adhesin protein, fractions thereof or said DNA isadministered in combination with a pharmaceutically suitableimmunostimulating agent. Examples of such agents include, but are notlimited to the following: cholera toxin and/or derivatives thereof, heatlabile toxins, such as E. coli toxin and similar agents. The compositionaccording to the present invention can further include conventional andpharmaceutically acceptable adjuvants, familiar to a person skilled inthe art of immunisation therapy. Preferably, in an immunisation therapyusing the inventive DNA or fractions thereof, said DNA is preferablyadministered intramuscularly, whereby said DNA is incorporated insuitable plasmide carriers. An additional gene or genes encoding asuitable immunostimulating agent can preferably be incorporated in thesame plasmide.

Said immunisation therapies are not restricted to the above-describedroutes of administration, but can naturally be adapted to any one of thefollowing routes of administration: oral, nasal, subcutaneous andintramuscular. Especially the oral and nasal methods of administrationare promising, in particular for large-scale immunisations.

The present inventors have surprisingly shown, that highly specific andtherapeutically efficient polyclonal and/or monoclonal immunoglobulinpreparations can be provided through the immunisation of an animal withan adhesin protein or fractions thereof, specific for H. pylori. Whenconsidering immunisation against H. pylori, it is worth noting that theinfection is known to be lifelong despite a vigorous immune response inthe gastric mucosa. An increased local production of IgA in the mucosais not necessarily enough and the administration of monospecificantibodies directed against a central virulens factor, such as theadhesin according to the present invention, may constitute a moreeffective approach.

The term “immunisation” refers here to a method for inducing acontinuous high level of antibody and/or cellular immunresponse. Theterm “animal” here preferentially denotes any member of the subphylumVertebrata, a division that includes all animals, including mammals,which are characterized by a segmented bony or cartilaginous spinalcolumn. All vertebrates have a functional immune system and respond toantigens by producing antibodies. The term “protein” is used here todenote a naturally occurring polypeptide and the term “polypeptide” isused here in its widest meaning, i.e. any amino acid polymer (dipeptideor longer) linked through peptide bonds. Accordingly the term“polypeptide” comprises proteins, oligopeptides, protein fragments,analogues, muteins, fusion proteins and the like. The term “antibody” asused in this context includes an antibody belonging to any of theimmunological classes, such as immunoglobulins A, D, E, G or M. Ofparticular interest are nevertheless immunoglobulin A (IgA) since thisis the principle immunoglobulin produced by the secretory system ofwarm-blooded animals. However, in cow colostrum, the main antibody classis IgG 1.

Borén et al. have recently isolated and characterized a Lewis^(b)binding protein with a molecular weight of about 73500 Da (See thepriority applications SE 9602287-6 and SE 9701014-4, which are referredto in their entirety). This adhesin protein is thought to be a conservedstructure and specific for pathogenic strains of H. pylori. Said proteinis specific for at least one of the H. pylori strains included in thefollowing group: CCUG 17875, NCTC 11637, A5, P466, G109, G56, Ba 185, Ba99, 931 and 932.

This adhesin protein or immunologically effective fractions thereof arecharacterized in that the following amino acid sequence (SEQ ID NO:5)included:

EDDGFYTSVGYQIGEAAQMV

or homologues thereof.

The following DNA sequence (SEQ ID NO:6) or homologues thereof isincluded in DNA for expression of said adhesin protein or fractionsthereof:

5′-GAAGACGACGGCTTTTACACAAGCGTAGGCTATCAAATCGGT GAAGCCGCTCAAATGGTA-3′

According to one embodiment of the invention, a pregnant mammal,preferably a cow or another suitable domestic animal, is immunised withsaid Lewis^(b) binding adhesin protein or fractions thereof. The adhesinprotein or fractions thereof is/are preferably injected intramuscularlyor subcutaneously in the chosen animal, optionally together withsuitable adjuvants. Examples of such adjuvants include, but are notlimited to immunostimulating agents such as cholera toxin and/orderivatives thereof, heat labile toxins, such as E. coli toxin andsimilar, conventional agents, such as classical adjuvants includingmineral and vegetable oils. Subsequent to the regimen of immunization,comprising a necessary amount of doses, including so calledbooster-doses, over a time span allowing for optimal immunoglobulinexpression, milk or sera is collected from said animal. Preferably thecow colostrum, which is specially high in immunoglobulins, is collected.The specific immunoglobulin fraction according to the present inventionis then separated and purified in a conventional manner, e.g. includingseparation of fats, protein precipitation and concentration byultrafiltration.

According to another embodiment of the invention, a bird, preferably achicken or another suitable domestic bird, is immunized with saidLewis^(b) binding adhesin protein or fractions thereof. The adhesinprotein or fractions thereof is preferably injected intramuscularly orsubcutaneously in the chosen bird, optionally together with suitableadjuvants. Examples of such adjuvants include, but are not limited toimmunostimulating agents such as cholera toxin and/or derivativesthereof, heat labile toxins, such as E. coli toxin and similar,conventional agents, such as classical adjuvants including mineral andvegetable oils. Subsequent to the regimen of inmmunization, comprising anecessary amount of doses, including so called booster-doses, over atime span allowing for optimal immunoglobulin expression, sera or eggsis/are collected from said animal. Preferably the egg yolk, which isspecially high in immunoglobulins, is collected. The specificimmunoglobulin fraction according to the present invention is thenseparated and purified in a conventional manner, e.g. including proteinprecipitation and ultrafiltration. Alternatively, the egg yolk being ofhigh nutritional value in addition to containing a high titer ofspecific antibodies according to the present invention, can beadministered as such.

According to a preferred embodiment of the present invention, monoclonalimmunoglobulin is produced by establishing transgenic animals. Saidtransgenic animals can be chosen from the following group of species:mammals, e.g. cow, goat and rabbit, and birds: e.g. chicken, duck,turkey. The mammal most preferably used is cow and the most preferablebird is chicken. Further developments of transgenic animals such as miceand rats could also offer new possibilities. The choice of animal isnaturally governed by availability and local adaptation.

According to one embodiment, a stock of transgenic animals according tothe present invention, adapted to the local conditions, are keptlocally, e.g. in villages in developing countries to function as localunits for the production of immunoglobulins for oral administration. Forexample transgenic cows, goats or chicken are suitable for this purposeand preferably chicken are used. Consumption of the milk or preferablythe eggs, produced by the transgenic animals, can help to eradicatepresently very difficult infectious diseases, e.g. diseases caused by H.pylori.

According to yet another embodiment of the present invention, monoclonalantibodies can be produced using the hybridoma method. The hybridomamethod is well known to a skilled worker in the field of biochemistryand it is described e.g. in Galfre, G. And Milstein, C., Preparation ofmonoclonal antibodies: strategies and procedures (Methods in Enzymology,73:3-46, 1981). A suitable host animal is immunized with the Lewis^(b)binding adhesin protein or fractions thereof. When the immunization isaccomplished, the animal is sacrificed, spleen cells collected and fusedwith cells from a neoplastic cell line, preferably myeloma cells. Bychoosing the growth conditions, the successfully fused hybridoma cellscan be selected. The monoclonal antibodies produced by the hybridomacell line can then be administered orally in a regimen for treatmentand/or prevention of H. pylori infections.

Preferably the polyclonal and/or monoclonal antibodies are purifiedprior to administration and, more preferably, admixed withpharmaceutically suitable carriers and/or adjuvants. Examples ofsuitable carriers are saline, pharmaceutically acceptable fats, oils,carbohydrates and proteins. The carrier or carriers is/are preferablychosen so that the solubility and absorption of the immunoglobulin inthe mucus layer lining the stomach is enhanced. Using suitable adjuvantsthe stability, therapeutic efficiency and nutritional value of thecomposition can be improved. To improve stability under storage, theimmnunoglobulin composition can be lyophilized. Regardless of the exactpreparation and formulation, it is of central importance to avoiddenaturating the immunoglobulins.

The higher specificity, exhibited by the immunoglobulin preparation ofpolyclonal and/or monoclonal antibodies according to the invention,makes it possible use substantially lower doses compared to thosepresently used, thus lowering the cost and improving the availability ofthe treatment. The use of specific, monoclonal antibodies can make itpossible to further lower the doses. The doses are in all cases afunction of the antibody titer of the preparation. A high titernaturally allows the use of lower doses.

According to one embodiment of the invention, an immunoglobulinpreparation is manufactured as follows: an animal is immunized with aLewis^(b) binding adhesin protein or fractions thereof, expressed byHelicobacter pylori, the immunoglobulin fraction is isolated from aexcretion of said animal and subsequently purified. The purifiedimmunoglobulin composition is admixed with suitable carriers andadjuvants to form a immunoglobulin preparation for the prevention ortreatment of H. pylori infections. In cases where the antibody titer issufficiently high and the other constituents of the immunoglobulincomposition isolated from the animal are harmless, for example in thecase of colostrum from immunized cows or egg yolk from immunizedchicken, there is always the option of administering the colostrum oregg yolk to the patient without any further treatment of the colostrumor egg yolk.

The immunoglobulin composition according to the invention is preferablyadministered orally to the patient, in the smallest therapeutically orprophylactically effective dose. Presently conceived are doses in theinterval of 0.1 to 1000 mg/day, preferably in the interval of 0.1 to 100mg/day. The chosen doses naturally depend on the antibody titer of thepreparation in question. The exact doses and the regimen ofadministration can be chosen by the physician responsible for thepatient, infected by Helicobacter pylori. Routine experimentation andlater, with increasing experience of this method, empirical informationwill suffice to establish the required amount. Multiple dosages may beused, as needed, to provide the desired level of therapeutic orprofylactic effect. The immunoglobulin preparations according to thepresent invention can also, being free from adverse side effects andimposing practically no danger of overdosing, be taken prophylacticallyor therapeutically by a person without medical supervision.

A therapeutical effect can be attained, except with the specificantibody according to the present invention, also with at least twoFab-fragments of said antibody. Said embodiment is also encompassed bythe scope of the present invention.

According to yet another embodiment, avirulent microorganisms,preferably bacteria, are used as expression systems for the specificantibody according to the present invention. An “avirulentmicroorganism” in this context is a microorganism which has the abilityto colonize and replicate in an infected individual, but which does notcause disease symptoms associated with virulent strains of the samespecies of microorganism. The definition inherent in the GRAS (GenerallyRegarded As Safe) concept can be applied here. A GRAS-organism issuitable for use according to the present invention, provided that theorganism externalises the antibody or can be modified to this effect.The term “microorganism” as used herein includes bacteria, protozoa andunicellular fungi. Preferably, bacteria are used as expression systems,e.g. bacteria of the genus Lactobacillus, Streptococcus orEnterobacteriae. The above mentioned expression system can be utilisedin vitro for the production of the specific antibody according to thepresent invention or, according to a further embodiment of theinvention, the micro-organism constituting the expression system can beadministered directly to the patient. The micro-organisms can beharvested and administered as such, but they are preferably mixed with asuitable carrier, mixed in a suitable foodstuff, lyophilised,encapsulated or treated in any other conventional way, used for thedelivery of viable micro-organisms to the gastrointestinal tract.

According to yet another embodiment, avirulent microorganisms,preferably bacteria, are used as expression systems for the specificadhesin protein according to the present invention. An “avirulentmicroorganism” in this context is a microorganism which has the abilityto colonize and replicate in an infected individual, but which does notcause disease symptoms associated with virulent strains of the samespecies of microorganism. The definition inherent in the GRAS (GenerallyRegarded As Safe) concept can be applied here. A GRAS-organism issuitable for use according to the present invention, provided that theorganism externalises the adhesin protein or can be modified to thiseffect. The term “microorganism” as used herein includes bacteria,protozoa and unicellular fungi. Preferably, bacteria are used asexpression systems, e.g. bacteria of the genus Lactobacillus,Streptococcus or Enterobacteriae. The above mentioned expression systemcan be utilised in vitro for the production of the specific adhesinaccording to the present invention or, according to a further embodimentof the invention, the micro-organism constituting the expression systemcan be administered directly to the patient. The micro-organisms can beharvested and administered as such, but they are preferably mixed with asuitable carrier, mixed in a suitable foodstuff, lyophilised,encapsulated or treated in any other conventional way, used for thedelivery of viable micro-organisms to the gastrointestinal tract.

The exact doses and the regimen of administration of saidmicro-organisms can be chosen by the physician responsible for thepatient, infected by Helicobacter pylori. Routine experimentation andlater, with increasing experience of this method, empirical informationwill suffice to establish the required amount. Multiple dosages may beused, as needed, to provide the desired level of therapeutic orprophylactic effect. The avirluent micro-organism expressing theantibody or adhesin protein according to the present invention can also,being free from adverse side effects and imposing practically no dangerof overdosing, be taken prophylactically or therapeutically by a personwithout medical supervision. A preferred carrier in this specificapplication is a foodstuff, e.g. a fermented product such as fermentedcereal or dairy product.

The creation of previously mentioned expression systems and stillearlier mentioned methods of creating hybridomas and transgenic animalscan include steps involving recombinant DNA techniques. Recombinant DNAtechniques are now sufficiently well known and widespread so as to beconsidered routine. In very general and broad terms, recombinant DNAtechniques consist of transferring part of the genetic material of oneorganism into a second organism, so that the transferred geneticmaterial becomes a permanent part of the genetic material of theorganism to which it is transferred. Methods for achieving this are wellknown and the mere choice of specific methods for achieving theobjectives, set out in the present description and claims, fall underthe scope of the invention.

It is possible, that H. pylori alone or together with relatedslow-acting bacteria are involved in the genesis and aggravation ofother chronic inflammatory diseases in the gastrointestinal tract. It isobvious for a skilled practitioner how to modify the present invention,within the scope of the claims, to gain utility in the treatment and/orprevention of such diseases. Examples of such diseases are ulcerativecolitis, Crohn's disease, sarcoidosis, Wegener's granulomatosis andother vasculithic disorders, as well as various neoplasms, includingcarcinomas of the colon, pancreas and prostate.

EXAMPLES

H. pylori strain CCUG 17875 was obtained from CCUG, Göteborg, Sweden.Strain A5, a gastric ulcer isolate, from Astra Arcus, Södertälje,Sweden. Strains P466 and MO19 were described previously (Borén et. al,Science, 262, 1892(1993)). Strain 26695 came from Dr. K. A. Eaton, TheOhio State University and its genome was recently sequenced by TIGR,Rockville, Md., USA. The panel of 45 H. pylori clinical isolates camefrom the University Hospital in Uppsala, Sweden. Bacteria were grown at37° C. in 10% CO2 and 5% O2 for 48 h.

All blood group antigen glycoconjugates used, i.e. semi-syntheticglycoproteins constructed by the conjugation of purified fucosylatedoligosacharides to serum albumin were from IsoSep AB, Tullinge, Sweden.The RIA was performed according to Falk et al. (Meth. Enzymol., 236,353, 1994) with some modifications; the H-1, Le^(b), Le^(a), H-2, Le^(x)and Le^(y) glycoconjugates were 125I-labeled by the Chloramine T method.1 ml of bacteria (A600=OD 0.10) was incubated with 300 ng of 125I-labelled conjugate (i.e. an excess of receptors) for 30 min. inphosphate buffered saline (PBS), 0.5% albumin, 0.05% Tween-20(BB-buffer). After centrifugation, 125I-activity in the bacterial pelletwas measured by gamma scintillation counting.

In this study the present inventors' first biochemically characterizedand identified the H. pylori blood group antigen binding adhesin, BabA.H. pylori strains were analysed for binding to soluble ¹²⁵I-labeledfucosylated blood group antigens (FIG. 1A). Binding of these strains tothe soluble blood group antigens correlate with adherence in situ. Theprevalence of blood group antigen binding (BAB)-activity was assessedamong 45 clinical H. pylori isolates and the majority of the isolates,71%, express Le^(b) antigen binding properties (data not shown). Incontrast, none of the reference strains (FIG. 1A), or strains from thepanel of 45 clinical isolates, bind to the Le^(a), H-2, Le^(x), orLe^(y) antigens. These results support our previous findings of highreceptor specificity for the Le^(b) and H-1 blood group antigens anddemonstrate the high prevalence of BAB activity among clinical isolates.

Based on the presence or absence of virulence factors such as theCytotoxin associated gene A (CagA) and the Vacuolating cytotoxin A(VacA), H. pylori strains are classified as type I or type II strains.H. pylori isolates from patients with duodenal ulcers most often expressthe VacA and the CagA-proteins, i.e. type-I strains. By definition, typeII strains express neither markers. Twenty-one clinical isolatespreviously defined for expression of CagA and VacA were analysed forLe^(b) antigen binding properties. Expression of CagA was found tocorrelate with bacterial binding to the Le^(b) antigen (Table 1). ThecagA gene belongs to a 40 kb pathogenicity island that encodescomponents of secretion and transport systems. These findings couldindicate functional crosstalk between the cag pathogenicity island andthe BabA adhesin gene, for the correct presentation of the BabA adhesinprotein in the bacterial outer membrane.

To further characterize BabA, the present inventors determined theaffinity constant (K_(a)) between BabA and the Le^(b) antigen. SinceK_(a)-values are based on equilibrium conditions (13), the presentinventors first analysed the interaction by performing receptordisplacement analysis. H. pylori CCUG 17875 (positive for Le^(b)binding, FIG. 1A) was first incubated with ¹²⁵I-labeled Le^(b)glycoconjugate. Then unlabeled Le^(b) glycoconjugate was added in adilution series. The unlabeled Le^(b) conjugate displaced the bound¹²⁵I-labeled Le^(b) glycoconjugate efficiently (FIG. 1B). The resultsdemonstrate that the receptor-adhesin complex formed is in a true stateof equilibrium. An equivalent excess of Le^(a) glycoconjugate did notdissociate the Le^(b)-BabA complex, verifying the high receptorspecificity (FIG. 1B). The K_(a)-value for the Le^(b)-BabA complex ofstrain CCUG 17875 was titrated with Le^(b) glycoconjugate inconcentrations from 10 ng to 260 ng/ml and determined to be of an highaffinity close to 1×10¹⁰M⁻¹ (FIG. IC). The number of Le^(b)glycoconjugate molecules bound to BabA on the bacterial cell surface wascalculated to be around 500 per cell. This number is similar to thenumber or fimbriae organelles on the surface of E. coli (14). However,for the BabA adhesin, the calculations are based on the assumption thatthe majority of bacterial cells in the experiment exhibit an equalnumber of adhesin molecules with Le^(b) antigen binding properties.

TABLE 1 BAB activity among H. pylori Type I and Type II strains TypeStrain BAB activity Type I CCUG 17874 − CagA⁺, VacA⁺ G39 − G11 − G20 −G27 + G56 + G106 − G109 + 932 + Ba185 + 87A300 + Type Ia 931 + CagA⁺,VacA⁻ Ba99 + Ba179 + Ba194 + Type Ib G12 − CagA⁻, VacA⁺ Type Id G104 −ΔcagA, VacA⁺ Tx30 − Type II G21 − CagA⁻, VacA⁻ G50 − G198 −

To determine the prevalence of BabA in the bacterial population, strainCCUG 17875 was incubated with Le^(b) or Le^(a) antigens, and bacterialbinding activity was visualised by confocal fluorescence microscopy(FIG. 2, upper panel). The analyses demonstrate the high prevalence ofBabA binding activity in the bacterial population to the Le^(b) antigen(FIG. 2A, green staining) and the complete lack of binding to the Le^(a)antigen (FIG. 2B, red counter staining).

Next, the localisation and density of BabA on the bacterial cellsurfaces was investigated by immunogold electron microscopy. The Le^(b)antigen binding activity of the adhesin localised gold particles to thebacterial outer membrane (FIG. 2C). Individual bacterial cells exhibitan equal number of gold particles (data not shown). When the Le^(b)antigen was substituted with the Le^(a) antigen (lacking receptoractivity), no gold particles were detected (FIG. 2D).

The molecular weight of BabA was characterized by receptor overlayanalysis. A protein extract of strain CCUG 17875 was separated onSDS-PAGE and blotted to a membrane. The membrane was incubated withbiotinylated Le^(b) glycoconjugate, followed by detection withstreptavidin and enhanced chemiluminescence. The BabA adhesin activitycorresponds to a single 74 kDa band (FIG. 3A). The 40 kDa band ispresumably endogenous peroxidase activity since it stains independentlyof the Le^(b) conjugate overlay (lane 3). BabA was very heat stable andcould regain some activity after heating to 97° C. (FIG. 3A, lane 2).The panel of strains exhibited the same molecular weight of BabA (FIG.3B).

To purify BabA, a novel technique was developed, Receptor ActivityDirected Affinity Tagging (ReTagging). Multi-functional crosslinkingagents with radiolabeled donating tags have been previously used forreceptor-ligand characterization studies. However, the use of affinitydonating tags, such as biotin residues presented on flexible spacerstructures, adds a new dimension to the applicability of crosslinkertechnology. An affinity tag, biotin, is transferred to the adhesinprotein by the receptor activity and is used for further identificationand for affinity purification of the adhesin part of the interaction, bystreptavidin (FIGS. 4A, B).

A multi-functional crosslinking agent with a biotin donating handle wasattached to the Le^(b) glycoconjugate. The receptor activity of theLe^(b) glycoconjugate subsequently directed the targeted biotin taggingof the BabA adhesin protein (FIGS. 4A, B). After crosslinking, thebacterial protein from strains A5, P466, and CCUG 17875 were separatedon SDS-PAGE. Immunodetection with streptavidin demonstrated a biotintagged protein, with the molecular weight of 74 kDa (FIG. 3C) (28),These results support the estimates of the molecular weight from theprevious overlay analyses (FIG. 3B). Strain MO19 devoid of Le^(b)antigen binding properties (FIG. 3B) (FIG. 1A), was negative for bindingalso in this set of analyses (FIG. 3C).

The high specificity in the ReTagging technique provided a method forpurification of the adhesin protein. Strains CCUG 17875 and A5, thatboth express the BabA adhesin (FIG. 1A) were processed by the ReTaggingtechnique using crosslinker labelled Le^(b) receptor conjugate as thebiotin donor. After crosslinking, bacteria were suspended in SDS samplebuffer. Streptavidin coated magnetic beads were subsequently added tothe solubilised proteins, and biotin tagged BabA was extracted (FIG.4C). The N-terminal 20 amino acid sequences of the BabA adhesins fromstrains CCUG 17875 (Australia) and A5 (Sweden) were found to beidentical, indicating a biologically conserved protein (FIG. 5).Recently, a series of outer membrane proteins from H. pylori werecharacterized. These proteins, HopA-E, are homologous in theirN-terminal sequences to BabA (17), possible indicating a motif for acommon secretion mechanism. The biotin tagged BabA adhesin was purifiedmore than 3000-fold from the cell extract, and the yield was calculatedto 20%. However, based on data from the Scatchard plots, the level ofavailable BabA adhesin would be about 5-times higher, i.e. approximately1 mg adhesin/750 mg bacterial protein, which nevertheless could be thereason for the high signal to noise ratio (FIG. 3B). The purification ofBabA via the ReTagging technique indicates the potential of thistechnique for the purification of lectins in complex receptor-ligandinteractions, such as the selectin family of cell adhesion molecules.

To clone the gene encoding BabA, the N-terminal 20 aa sequence wasutilised for the construction of degenerate primers (18). Two sets ofclones were identified which both encode two different but very similarproteins. Both genes code for proteins having almost identicalN-terminal domains and identical C-terminal domains, complicating theidentification of the functional BabA gene. (FIG. 5). To identify thecorresponding gene, the BabA adhesin was purified in large scale byReTagging. This provided enough protein for an extended amino terminalsequence. 41 amino acids were identified and these residuesunambiguously discriminated between the two genes by the differences inaa-positions 28, 35, 37, 38 and 41 (FIG. 5). The gene encoding BabA wasnamed babA and correspond to a basic protein with a pI of 9.4 and amolecular weight of 78 kDa, i.e. of slightly higher molecular weightthan that predicted from the SDS PAGE analyses (FIG. 3). The other gene,babB, corresponds to a protein of a calculated molecular weight of 75.5kDa. In contrast to babA, the babB gene contains a predictedtranslational initiation codon (FIG. 5). This could indicate theexistence of a third bab gene in the genome or mechanisms forrecombination activities. Interestingly, the bab-genes were alsodetected in strains lacking Lewis b binding properties (data not shown).Gene cassette systems have been shown to promote antigenic variation inNeisseria gonorrhoeae (19). Another possibility would be the presence ofsimilar genes coding for adhesins with differences in receptorspecificity/host tissue tropism (20). Gene inactivation experimentstargeting the bab-genes could aid in understanding this complex geneorganisation.

Immunisation experiments with adhesins from Bordetella pertussis (21)indicate the potential for outer membrane proteins to act as vaccinecandidates (discussed in ref 22). In a mouse model for persistent H.pylori infection, oral immunisation with H. pylori antigens provedprotective against H. pylori infection (10). However, results fromanimal models are difficult to evaluate for human specific pathogens,such as H. pylori and Polio virus. For Polio, an animal model has beenachieved by expressing the virus receptor in transgenic mice (23). Asimilar strategy was taken for H. pylori. A transgenic mouse wasconstructed by the use of an a1,3/4-fucosyltransferase, driving thesynthesis of the human specific Le^(b) antigen in the gastrointestinaltract (24). The Lewis b mouse can be useful for the evaluation of therole of the BabA adhesin as a colonisation/virulence factor and inaddition for the evaluation of BabA as a vaccine candidate against acidpeptic disease and gastric adenocarcinoma.

In the present study the ReTagging technique was used for thepurification of the adhesin part of the microbial receptor-ligandinteraction. By the use of purified adhesin/lectin-protein, theReTagging technique could, in addition, be used to further study thereceptor part of the interaction. Identification of the biologicallyactive receptor structure, carrying Le^(b) oligosaccharides, would aidin the understanding of the mechanisms supporting the chronic H. pyloriinfection.

Inhibition of H. pylori binding to ¹²⁵I-labeled Lewis b antigen bypreparations is presented graphically, as a function of antibodyconcentration (mg/ml) in FIG. 6: 1 ml aliquots of H. pylori bacteria(A₆₀₀=OD 0.10) were pre-incubated with dilution series of antibodypreparations, in 0.01-10 mg/ml for 2 hours in phosphate buffered saline(PBS), 0.5% albumin, 0.05% Tween-20™. Then 500 ng of ¹²⁵I-labeledconjugate (i.e. an excess of receptor structure) was added and incubatedfor 30 minutes. After centrifugation, ¹²⁵I-activity in the bacterialpellet was measured by gamma scintillation counting. The Lewis b bloodgroup antigen glycoconjugates used, i.e. semi-synthetic glycoproteinsconstructed by the conjugation of purified fucosylated oligosaccharidesto serum albumin were from IsoSep AB, Tullinge, Sweden.

Western blot detection of the BabA adhesin by the different antibodypreparations is presented in FIG. 7: Molecular weight rainbow marker (2μL) from Amersham, Buckinghamshire, England, was dissolved in SDS samplebuffer (lane 1). Approx. 100 ng of purified BabA adhesin (approx. 74 kDawith degradation product of approx. 55 kDa) was dissolved in SDS samplebuffer (lane 2). SDS solubilized protein extracts of strain CCUG 17875were prepared by dissolving the bacterial pellet corresponding to 0.15ml of bacteria (A₆₀₀=OD 0.10) by SDS sample buffer (lane 3). The 3protein samples were then boiled at 100° C. for 5 minutes. The proteinswere separated on SDS-PAGE, and transferred to a PVDF-membrane forWestern blot immuno analysis. Five sets of PVDF-membranes were prepared.The PVDF membranes were blocked/incubated overnight with 4% humansera/plasma, in phosphate buffered saline, from a patient with no H.pylori infection, i.e. with no serum antibodies against H. pylori. Themembrane was then washed in phosphate buffered saline (PBS), 0.5%albumin, 0.05% Tween-20, followed by the addition of the antibodypreparations. The sets of membranes were incubated with the following 5antibody preparations; 1) pooled human sera from H. pylori infectedpatients, diluted 1:500. 2) Chicken antibodies (positive) 1 mg/mldiluted 1:100×, 3) Bovine I preparation of antibodies, 1 mg/ml diluted1:100×. 4) Bovine II preparation of antibodies, 1 mg/ml diluted 1:100×.5) Bovine III preparation of antibodies, 1 mg/ml diluted 1:100×(indicated in the figure). These antibodies were incubated with themembrane for 2 hours followed by extensive washings in phosphatebuffered saline (PBS), 0.05% Tween-20, followed by the addition ofsecondary anti-human, anti-chicken, and anti-bovine antibodies labeledwith HRP-peroxidase (from DAKO, Denmark), all diluted 1:2000×. Membraneswere incubated for 1 hour, followed by extensive washings in phosphatebuffered saline (PBS), 0.05% Tween-20. The membranes were developed withenhanced chemoluminescens (ECL) from Amersham. The results show, thatthe antigenic response against the adhesin is strongly enchanced in thebovine preparations. This finding is also supported by the inhibitiondata in FIG. 6.

Western blot analyses of H. pylori proteins by the different antibodypreparations are shown in FIG. 8. 2 clinical isolates (1-2) from Dr.Lars Engstrand, Department of Clinical Microbiology andCancerepidemiology, University Hospital, Uppsala, Sweden and strain CCUG17875 (3), from Culture Collection, University of Göteborg, Departmentof Clinical Bacteriology, Göteborg, Sweden, and strain 52 (4) from Prof.Torkel Wadström, Dept. Medical Microbiology, Lunds University, wereprepared for SDS-PAGE electrophoresis. Bacterial pellets correspondingto 0.15 ml of bacteria (A₆₀₀=OD 0.10) were dissolved in SDS samplebuffer and heated to 100° C. for 5 minutes. The proteins were separatedon SDS-PAGE, and transferred to PVDF-membranes for Western blot immunoanalysis. The western blot analyses were as described above, i.e. thesets of membranes were incubated with the following 4 antibodypreparations; 1) pooled human sera from H. pylori infected patients,diluted 1:500. 2) Chicken antibodies (positive) 1 mg/ml diluted 1:100×,3) Bovine I preparation of antibodies, 1 mg/ml diluted 1:100×. 4) BovineIII preparation of antibodies, 1 mg/ml diluted 1:100× (indicated in thefigure). These antibodies were incubated with the membrane for 2 hoursfollowed by extensive washings in phosphate buffered saline (PBS), 0.05%Tween-20, followed by the addition of secondary anti-human,anti-chicken, and anti-bovine antibodies labeled with HRP-peroxidase(from DAKO, Denmark), all diluted 1:2000×. Membranes were incubated for1 hour, followed by extensive washings in phosphate buffered saline(PBS), 0.05% Tween-20. The membranes were developed with enhancedchemoluminescens (ECL) from Amersham. The results show, that the chickenantibodies and the bovine preparations reacts nearly identically againstall four strains, indicating conserved properties in strains ofdifferent geographical origin.

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventors, it should be understood that various changes andmodifications as would be obvious to one having the ordinary skill inthis art may be made without departing from the scope of the inventionwhich is set forth in the claims appended hereto.

REFERENCES AND NOTES

1. J. R. Warren, Lancet, i, 1273, 1983, B. Marshall, Lancet, i, 1273,1983.

2. A. Dubois, Emerging Infectious Diseases 1, 79 (1995).

3. M. J. Blaser, Sci. Amer. 2, 92 (1996).

4. 3. M. J. Blaser, Trends Microbiol. 7, 255 (1993), D. E. Kirschner andM. J. Blaser, J. Theor. Biol. 176, 281 (1995).

5. P. Falk, T. Borén, and S. Normark, Meth. Enzymol. 236, 353 (1994).

6. D. G. Evans, D. J. Evans Jr., J. J. Moulds and D. Y. Graham, Infect.Immun. 56, 2896 (1988), S. Hirmo, M. Utt, M. Ringner and T. Wadström,FEMS Immunol. and Med Microbiol. 10, 301 (1995), T. Saitoh, et al FEBSLett. 282, 385 (1991).

7. T. Borén, P. Falk, K. A. Roth, G. Larson, and S. Normark, Science.262, 1892 (1993), P. Falk, et al Proc. Natl. Acad. Sci. U.S.A. 90, 2035(1993).

8. H. pylori strain CCUG 17875 was obtained from CCUG, Göteborg, Sweden.Strain A5, a gastric ulcer isolate, came from Astra Arcus, Södertälje,Sweden. Strains P466 and MO19 were described previously (7). Strain26695 came from Dr. K A. Eaton, The Ohio State University, and itsgenome was recently sequenced by The Institute for Genomic Research(TIGR), Rockville, Md. (J.-F. Tomb, et al, abstract 3B: 059, IXInternational Workshop on Gastroduodenal Pathology and Helicobacterpylori, Copenhagen, Denmark, 1996). The panel of 45 H. pylori clinicalisolates came from the University Hospital in Uppsala, Sweden. Bacteriawere grown at 37(C. in 10% CO₂ and 5% O₂ for 48 h.

9. All blood group antigen glycoconjugates used, i.e. semi-syntheticglycoproteins constructed by the conjugation of purified fucosylatedoligosaccharides to serum albumin (7, 25), were from IsoSep AB,Tullinge, Sweden. The RIA was performed according to ref. 26 with somemodifications; The H-1, Le^(b), Le^(a), H-2, Le^(x), and Le^(y)glycoconjugates were ¹²⁵I-labeled by the Chloramine T method. 1 ml ofbacteria (A₆₀₀=OD 0.10) was incubated with 300 ng of ¹²⁵I-labeledconjugate (i.e. an excess of receptors) for 30 min. in phosphatebuffered saline (PBS), 0.5% albumin, 0.05% Tween-20 (BB-buffer). Aftercentrifugation, ¹²⁵I-activity in the bacterial pellet was measured bygamma scintillation counting.

10. A. Covacci, et al, Proc. Natl. Acad. Sci. U.S.A. 90, 5791 (1993), M.Marchetti, et al, Science 267, 1655 (1995).

11. S. Censini et al, Proc. Natl. Acad. Sci. U.S.A. 93, 14648, (1996).

12. Z. Xiang, et al, Infect. Immun. 63, 94 (1995).

13. A. G. Scatchard, Ann. N. Y. Acad. Sci. 51, 600 (1949).

14. O. Mol, and B. Oudega, FEMS. Microbiol. Reviews, 19, 25 (1996).

15. Confocal microscopy was performed on a Nikon/Multiprobe 2001instrument (Molecular Dynamics, Sunnyvale, Calif.). Electron microscopywas performed on a JEOL 100 CX instrument.

16. J. Brunner, Trends in Cell Biol. 6, 154 (1996), J. D. Bleil and P.M. Wassarman, Proc. Natl. Acad. Sci. U.S.A. 87, 5563, (1990).

17. M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect.Immun. 63, 1567 (1995), P. Doig, M. M. Exner, R. E. W. Hancock and T. J.Trust, J. Bacteriol. 177, 5447 (1995).

18. The BabA N-terminal sequence analysis was used to make degenerateoligonucleotides, which were used in PCR to obtain an amplified fragmentfrom the chromosome of the baba gene. A 59 bp fragment was identifiedand used as probe for the screening of a low-copy plasmide (pACYC184)library of Sau3A partially digested chromosomal DNA from strain CCUG17875.

19. P. Hagblom, E. Segal, E. Billyard, and M. So, Nature, 315, 156(1985), R. Haas and T. F. Meyer, Cell, 44, 107 (1986).

20. A.-B. Jonsson, D. Ilver, P. Falk, J. Pepose, and S. Normark, Mol.Microbiol, 13, 403 (1994), N. Strömberg, P. G. Nyholm, I. Pascher, andS. Normark, Proc. Nail. Acad. Sci USA 88, 9340 (1991).

21. A. Kimura, K. T. Mountzouros, D. A. Relman, S. Falkow, J. L. Cowell,Infect. Immun. 58, 7 (1990).

22. T. Borén, and P. Falk, Sci. Amer., Sci. & Med. 4 (1994), L. S.Tompkins and S. Falkow, Science 267, 1621 (1995).

23. R. B. Ren, et al, Cell 63, 353 (1990).

24. P. G. Falk, L. Bry, J. Holgersson, and J. I. Gordon, Proc. Natl.Acad. Sci. U.S.A. 92, 1515 (1995).

25. P. D. Rye, Nature Biotechnology. 2, 155 (1996).

26. P. Falk, T. Borén, D. Haslam, M. G. Caparon, Meth. Cell Biol. 45,161 (1994)

27. Cell extracts were prepared in SDS sample buffer without mercaptoethanol and heated at 37° C. or 97° C. for 10 min. before separation onSDS-PAGE. Proteins were blotted onto a PVDF membrane. The membrane wasincubated with 1 μg/ml biotinylated Le^(b) glycoconjugate orbiotinylated albumin (negative control) overnight, labelled as describedin ref 7. After washing in PBS/0.05% Tween-20, the biotinylatedstructures bound by the BabA band were probed by HRP-streptavidin anddetected using ECL reagents (Amersham, Buckinghamshire, England).

28. The bacterial suspension was incubated with Le^(b) glycoconjugate,to which the Sulfo-SBED crosslinker (Pierce, Rockville, Ill.) had beenconjugated by the N-hydroxysuccinimide ester (NHS), according to themanufacturers specifications. The aryl azide crosslinker group wasactivated by UV irradiation (360 nm). Bacteria were washed with PBS pH7.6, 0.05% Tween-20 and protease inhibitors (EDTA and benzamidine) underreducing conditions with 50 mM dithiothreitol (DTT). Bacterial proteinswere separated on SDS-PAGE, and the biotin tagged BabA protein wasdetected by immunodetection (PVDF membrane/HRP-streptavidin and ECL)(FIG. 3C).

29. Strains CCUG 17875 and A5 were first processed by crosslinking andDTT treatment, as above (28), followed by solubilisation in SDS samplebuffer. The biotin tagged BabA protein was then extracted withstreptavidin coated magnetic beads (Advanced Magnetics Inc., Cambridge,Mass.). The beads were boiled in SDS sample buffer, and bound proteinswere eluted and alkylated. The protein preparation was furtherfractionated by preparative SDS-PAGE (Prep-Cell 491, BioRad, Hercules,Calif.). Fractions with the biotin tagged protein, i.e. the BabAfractions, were identified by immunodetection using streptavidin/ECL.The pooled BabA preparation was then separated on SDS-PAGE andtransferred to PVDF membrane. The BabA band was excised and the BabAprotein was N-terminally sequenced using a Procise™ 494 instrument(Applied Biosystems, Foster City, Calif.).

8 1 3340 DNA Helicobacter pylori 1 tttcagtcaa gcccaaagct atgcgcaaaacgcttatgct aaagagaatt tacaagcaca 60 gccgtccaag tatcaaaaca gcgtgcctgaaatcaatatt gatgaagaag aaatcccctt 120 taagggttaa aattaaggag acattatggaaagaaaacgc tattcaaaac gctattgcaa 180 atacactgaa gctaaaatca gctttattgactataaagat ttggacatgc tcaagcacac 240 gctatcagag cgctataaaa tcatgccaaggaggttgaca ggcaatagca aaaagtggca 300 agagagggtg gaagttagcg atcaaaagagcccgccacat ggctttaatc ccctacattg 360 tggataggaa aaaagtcgtg gatagcccttttaaacagca ctgaattttt gattagggct 420 aatagggggc atgcctttta atcttgtttaatcttggctc tatttttgtt aaacatcggt 480 tataaaagcg ttaaaagcac ttttaaaatccaattaaaag cgttcaaaag taacgcaaaa 540 aatcaaaaaa atgacaaaat ttttaagaaaatgacaaaaa aaaaaaaaac gctttatgct 600 ataatattcc aaatacattc taatgcaaatgcattctaat gcaaatgtat aatgaatgta 660 tgaaatccct aatattcaat ccaatttaatccaaaaagga gaaaaaacac atcctttcat 720 taactttagg ctcgctttta gtttccactttgagcgctga agacgacggc ttttacacaa 780 gcgtaggcta tcaaatcggt gaagccgctcaaatggtaac aaacaccaaa ggcatccaag 840 atctttcaga caactatgaa aacttgagcaaacttttgac ccgatacagc accctaaaca 900 cccttatcaa attgtccgct gatccgagcgcgattaacgc ggcacgtgaa aatctgggcg 960 cgagcgcgaa gaacttgatc ggcgataccaaaaattcccc cgcctatcaa gccgtgcttt 1020 tggcgatcaa tgcggcggta gggttttggaatgtcttagg ctatgctacg caatgcgggg 1080 gtaacgctaa tggtcaagaa agcacctcttcaaccaccat cttcaacaac gagccagggt 1140 atcgatccac ttccatcact tgcagtttgaacaggtataa gcctggatac tacggcccta 1200 tgagcattga aaatttcaaa aagcttaacgaagcctatca aatcctccaa acggctttaa 1260 ataaaggctt acccgcgctc aaagaaaacaacggaacggt cagtgtaacc tacacctaca 1320 catgctcagg ggaagggaat gataactgctcgaaaaaagc cacaggtgta agtgaccaaa 1380 atggcggaac caaaactaaa acccaaaccatagacggcaa aaccgtaacc accacgatca 1440 gttcaaaagt cgttgatagt caggcaaaaggtaatacaac aagggtgtcc tacaccgaaa 1500 tcactaacaa attagacggt gtgcctgatagcgctcaagc gctcttggcg caagcgagca 1560 cgctcatcaa caccatcaac acggcatgcccgtattttag tgtaactaat aaaagtggtg 1620 gtccacagat ggaaccgact agagggaagttgtgcggttt tacagaagaa atcagcgcga 1680 tccaaaagat gatcacagac gcgcaagagctggtcaatca aacgagcgtc attaacgagc 1740 atgaacaatc aaccccggta ggcggtaataatggcaagcc tttcaaccct ttcacggacg 1800 caagcttcgc tcaaggcatg ctcgctaacgctagtgcgca agccaaaatg ctcaatctag 1860 cccatcaagt ggggcaaacc attaaccctgacaatcttac cgggactttt aaaaattttg 1920 ttacaggctt tttagccaca tgcaacaacaaatcaacagc tggcactagt ggcacacaag 1980 gttcacctcc tggcacagta accactcaaactttcgcttc cggttgcgcc tatgtggagc 2040 aaaccataac gaatctaaac aacagcatcgctcattttgg cactcaagag cagcagatac 2100 agcaagctga aaacatcgct gacactctagtgaatttcaa atctagatac agcgaattag 2160 ggaatactta taacagcatc actactgcgctctccaaagt ccctaacgcg caaagcttgc 2220 aaaacgtggt gggaaaaaag aataacccctatagcccgca aggcatagaa accaattact 2280 acttgaatca aaactcttac aaccaaatccaaaccatcaa ccaagaatta gggcgtaacc 2340 cctttaggaa agtgggcatc gtcagttctcaaaccaacaa tggtgccatg aatgggatcg 2400 gtatccaggt gggctacaag caattctttgggcaaaaaag gaaatggggt gcaagatact 2460 acggcttttt tgattacaac catgcgttcattaaatccag cttcttcaac tcggcttctg 2520 acgtgtggac ttatggtttt ggagcggacgctctttataa cttcatcaac gataaagcca 2580 ccaatttctt aggcaaaaac aacaagctttctgtggggct ttttggcggg attgcgttag 2640 cgggcacttc atggcttaat tctgaatacgtgaatttagc caccatgaat aacgtctata 2700 acgctaaaat gaacgtggcg aacttccaattcttattcaa catgggagtg aggatgaatt 2760 tagccagatc caagaaaaaa ggcagcgatcatgcggctca gcatggcatt gagttagggc 2820 ttaaaatccc caccattaac acgaactactattcctttat gggggctgaa ctcaaatacc 2880 gcaggctcta tagcgtgtat ttgaattatgtgttcgctta ctaaaaacta aaaatccttt 2940 gtggaactcc ctttttaagg ggtttcttttaaagccttta tttttttttg gaggggttta 3000 atttttttga aacctttgtt tttgaattctctttttaatg ggtttctttt ttgaactctt 3060 tgttttgaac tccttttttt gaactcccttttttaaaccc tttctttttt aaaattctct 3120 tttttggggg gtttgatgaa aaatccttttttagcgtttt ggtattggtt agtggaaaac 3180 ttgatactaa tttaagcgat agtttttaaaaagtgcttct ttaatatagg gggtttaagt 3240 tggtgattaa aaggggggaa tggtttcaaagcgcttccta tccctttaag aaaataaaat 3300 aaaactttaa taaaatgagt tttacaacaaaatgagatcc 3340 2 2781 DNA Helicobacter pylori 2 catttgatcg cattggatttcaaagaaggg cgttttgtga aaggctttgg tcaagcttat 60 gatattttag gcgacaaaatcgcttatgtt gggggtaaag gcaacccaca caatttcgct 120 cacaagaaat aaactttctcacccataagg ggcaaacgcc cccaaaagag tgctttttaa 180 agaggttaag gcaaaatcaagctctttagt atttaatctt aaaaaatact aaaagccttt 240 ttatgggcta acaccacacaaaaagcgtca aaatcaaaaa aatgacaaaa ttttccccaa 300 atgacaaaaa aaaaaaaaaacgattttatg ctatattaac gaaatcttgt gataagatct 360 tattctttta aaagatttacctaaccattt taatttcaag gagaaaacat gaaaaaaaac 420 ccttttactc tctctctctctctctcgttt ttgctccacg ctgaagacga cggcttttac 480 acaagcgtag gctatcaaatcggtgaagcc gctcaaatgg taaccaacac caaaggcatc 540 caacagcttt cagacaattatgaaaagctg aacaatcttt tgaataatta cagcacccta 600 aacaccctta tcaaattatccgctgatccg agtgcgatta acgacgcaag ggataatcta 660 ggctcaagtg ctaagaatttgcttgatgtt aaaaccaact ccccggccta tcaagccgtg 720 cttttagcgt tgaatgcggcggtggggttg tggcaagtta caagctacgc ttttactgct 780 tgtggtcctg gcagtaacgagagcgcaaat ggaggtatcc aaacttttaa taatgtgcca 840 ggacaaaaga cgacaaccatcacttgcaat tcgtattatc aaccaggaca tggtgggcct 900 atatccactg caaactatgcaaaaatcaat caagcctatc aaatcattca aaaggctttg 960 acagccaatg aagctaatggagatggggtc cccgttttaa gcgacaccac tacaaaactt 1020 gatttcacta ttcaaggagacaaaagaacg ggtggccgac caaatacacc taaaaagttc 1080 ccatggagtg atgggaaatatattcacacc caatggattg acacaacacc acaatcaaca 1140 gaaacaaaga tcaacacagaaaataacgct caagagcttt taaaacaagc gagcatcatt 1200 atcactaccc taaatgaggcatgcccaaac ttccaaaatg gtggtagcgg ttattggcaa 1260 gggataagcg gcaatgggacaatgtgtggg atgtttaaga atgaaatcag cgctatccaa 1320 ggcatgatcg ctaacgcgcaagaagctgtc gcgcaaagta aaatcgttag tgaaaatgcg 1380 caaaatcaaa acaacttggatactggaaaa ccattcaacc ctttcacgga cgctagcttc 1440 gctcaaagca tgctcaaaaacgctcaagcc caagcagaga ttttaaacca agccgaacaa 1500 gtggtgaaaa actttgaaaaaatccctaaa aattttgtat cagactcttt aggggtgtgt 1560 tatgaagagc aagggggtgagcgtaggggc accaatccag gtcaggttac ttctaacact 1620 ttcgcttccg gttgcgcctatgtggagcaa accataacga atctaaacaa cagcatcgct 1680 cattttggca ctcaagagcagcagatacag caagctgaaa acatcgctga cactctagtg 1740 aatttcaaat ctagatacagcgaattaggg aatacttata acagcatcac tactgcgctc 1800 tccaaagtcc ctaacgcgcaaagcttgcaa aacgtggtgg gaaaaaagaa taacccctat 1860 agcccgcaag gcatagaaaccaattactac ttgaatcaaa actcttacaa ccaaatccaa 1920 accatcaacc aagaattagggcgtaacccc tttaggaaag tgggcatcgt cagttctcaa 1980 accaacaatg gtgccatgaatgggatcggt atccaggtgg gctacaagca attctttggg 2040 caaaaaagga aatggggtgcaagatactac ggcttttttg attacaacca tgcgttcatt 2100 aaatccagct tcttcaactcggcttctgac gtgtggactt atggttttgg agcggacgct 2160 ctttataact tcatcaacgataaagccacc aatttcttag gcaaaaacaa caagctttct 2220 gtggggcttt ttggcgggattgcgttagcg ggcacttcat ggcttaattc tgaatacgtg 2280 aatttagcca ccatgaataacgtctataac gctaaaatga acgtggcgaa cttccaattc 2340 ttattcaaca tgggagtgaggatgaattta gccagatcca agaaaaaagg cagcgatcat 2400 gcggctcagc atggcattgagttagggctt aaaatcccca ccattaacac gaactactat 2460 tcctttatgg gggctgaactcaaataccgc aggctctata gcgtgtattt gaattatgtg 2520 ttcgcttact agaaactaaaaatcctttgt ggaactccct ttttaagggg tttcttttaa 2580 agcctttatt tttttttggaggggtttaat ttttttgaaa cctttgtttt tgaattctct 2640 ttttaatggg tttcttttttgaactctttg ttttgaactc ctttttttga actccctttt 2700 ttaaaccctt tcttttttaaaattctcttt tttggggggt ttgatgaaaa atcctttttt 2760 agcgttttgg tattggttag t2781 3 744 PRT Helicobacter pylori PEPTIDE (24)..(64) N-terminal domainof the BabA adhesin 3 Ser Lys Lys Glu Lys Lys His Ile Leu Ser Leu ThrLeu Gly Ser Leu 1 5 10 15 Leu Val Ser Thr Leu Ser Ala Glu Asp Asp GlyPhe Tyr Thr Ser Val 20 25 30 Gly Tyr Gln Ile Gly Glu Ala Ala Gln Met ValThr Asn Thr Lys Gly 35 40 45 Ile Gln Asp Leu Ser Asp Asn Tyr Glu Asn LeuSer Lys Leu Leu Thr 50 55 60 Arg Tyr Ser Thr Leu Asn Thr Leu Ile Lys LeuSer Ala Asp Pro Ser 65 70 75 80 Ala Ile Asn Ala Ala Arg Glu Asn Leu GlyAla Ser Ala Lys Asn Leu 85 90 95 Ile Gly Asp Thr Lys Asn Ser Pro Ala TyrGln Ala Val Leu Leu Ala 100 105 110 Ile Asn Ala Ala Val Gly Phe Trp AsnVal Leu Gly Tyr Ala Thr Gln 115 120 125 Cys Gly Gly Asn Ala Asn Gly GlnGlu Ser Thr Ser Ser Thr Thr Ile 130 135 140 Phe Asn Asn Glu Pro Gly TyrArg Ser Thr Ser Ile Thr Cys Ser Leu 145 150 155 160 Asn Arg Tyr Lys ProGly Tyr Tyr Gly Pro Met Ser Ile Glu Asn Phe 165 170 175 Lys Lys Leu AsnGlu Ala Tyr Gln Ile Leu Gln Thr Ala Leu Asn Lys 180 185 190 Gly Leu ProAla Leu Lys Glu Asn Asn Gly Thr Val Ser Val Thr Tyr 195 200 205 Thr TyrThr Cys Ser Gly Glu Gly Asn Asp Asn Cys Ser Lys Lys Ala 210 215 220 ThrGly Val Ser Asp Gln Asn Gly Gly Thr Lys Thr Lys Thr Gln Thr 225 230 235240 Ile Asp Gly Lys Thr Val Thr Thr Thr Ile Ser Ser Lys Val Val Asp 245250 255 Ser Gln Ala Lys Gly Asn Thr Thr Arg Val Ser Tyr Thr Glu Ile Thr260 265 270 Asn Lys Leu Asp Gly Val Pro Asp Ser Ala Gln Ala Leu Leu AlaGln 275 280 285 Ala Ser Thr Leu Ile Asn Thr Ile Asn Thr Ala Cys Pro TyrPhe Ser 290 295 300 Val Thr Asn Lys Ser Gly Gly Pro Gln Met Glu Pro ThrArg Gly Lys 305 310 315 320 Leu Cys Gly Phe Thr Glu Glu Ile Ser Ala IleGln Lys Met Ile Thr 325 330 335 Asp Ala Gln Glu Leu Val Asn Gln Thr SerVal Ile Asn Glu His Glu 340 345 350 Gln Ser Thr Pro Val Gly Gly Asn AsnGly Lys Pro Phe Asn Pro Phe 355 360 365 Thr Asp Ala Ser Phe Ala Gln GlyMet Leu Ala Asn Ala Ser Ala Gln 370 375 380 Ala Lys Met Leu Asn Leu AlaHis Gln Val Gly Gln Thr Ile Asn Pro 385 390 395 400 Asp Asn Leu Thr GlyThr Phe Lys Asn Phe Val Thr Gly Phe Leu Ala 405 410 415 Thr Cys Asn AsnLys Ser Thr Ala Gly Thr Ser Gly Thr Gln Gly Ser 420 425 430 Pro Pro GlyThr Val Thr Thr Gln Thr Phe Ala Ser Gly Cys Ala Tyr 435 440 445 Val GluGln Thr Ile Thr Asn Leu Asn Asn Ser Ile Ala His Phe Gly 450 455 460 ThrGln Glu Gln Gln Ile Gln Gln Ala Glu Asn Ile Ala Asp Thr Leu 465 470 475480 Val Asn Phe Lys Ser Arg Tyr Ser Glu Leu Gly Asn Thr Tyr Asn Ser 485490 495 Ile Thr Thr Ala Leu Ser Lys Val Pro Asn Ala Gln Ser Leu Gln Asn500 505 510 Val Val Gly Lys Lys Asn Asn Pro Tyr Ser Pro Gln Gly Ile GluThr 515 520 525 Asn Tyr Tyr Leu Asn Gln Asn Ser Tyr Asn Gln Ile Gln ThrIle Asn 530 535 540 Gln Glu Leu Gly Arg Asn Pro Phe Arg Lys Val Gly IleVal Ser Ser 545 550 555 560 Gln Thr Asn Asn Gly Ala Met Asn Gly Ile GlyIle Gln Val Gly Tyr 565 570 575 Lys Gln Phe Phe Gly Gln Lys Arg Lys TrpGly Ala Arg Tyr Tyr Gly 580 585 590 Phe Phe Asp Tyr Asn His Ala Phe IleLys Ser Ser Phe Phe Asn Ser 595 600 605 Ala Ser Asp Val Trp Thr Tyr GlyPhe Gly Ala Asp Ala Leu Tyr Asn 610 615 620 Phe Ile Asn Asp Lys Ala ThrAsn Phe Leu Gly Lys Asn Asn Lys Leu 625 630 635 640 Ser Val Gly Leu PheGly Gly Ile Ala Leu Ala Gly Thr Ser Trp Leu 645 650 655 Asn Ser Glu TyrVal Asn Leu Ala Thr Met Asn Asn Val Tyr Asn Ala 660 665 670 Lys Met AsnVal Ala Asn Phe Gln Phe Leu Phe Asn Met Gly Val Arg 675 680 685 Met AsnLeu Ala Arg Ser Lys Lys Lys Gly Ser Asp His Ala Ala Gln 690 695 700 HisGly Ile Glu Leu Gly Leu Lys Ile Pro Thr Ile Asn Thr Asn Tyr 705 710 715720 Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr Arg Arg Leu Tyr Ser Val 725730 735 Tyr Leu Asn Tyr Val Phe Ala Tyr 740 4 707 PRT Helicobacterpylori PEPTIDE (46)..(59) Corresponding to the N-terminal domain of theBabA adhesin 4 Met Lys Lys Asn Pro Phe Thr Leu Ser Leu Ser Leu Ser PheLeu Leu 1 5 10 15 His Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly TyrGln Ile Gly 20 25 30 Glu Ala Ala Gln Met Val Thr Asn Thr Lys Gly Ile GlnGln Leu Ser 35 40 45 Asp Asn Tyr Glu Lys Leu Asn Asn Leu Leu Asn Asn TyrSer Thr Leu 50 55 60 Asn Thr Leu Ile Lys Leu Ser Ala Asp Pro Ser Ala IleAsn Asp Ala 65 70 75 80 Arg Asp Asn Leu Gly Ser Ser Ala Lys Asn Leu LeuAsp Val Lys Thr 85 90 95 Asn Ser Pro Ala Tyr Gln Ala Val Leu Leu Ala LeuAsn Ala Ala Val 100 105 110 Gly Leu Trp Gln Val Thr Ser Tyr Ala Phe ThrAla Cys Gly Pro Gly 115 120 125 Ser Asn Glu Ser Ala Asn Gly Gly Ile GlnThr Phe Asn Asn Val Pro 130 135 140 Gly Gln Lys Thr Thr Thr Ile Thr CysAsn Ser Tyr Tyr Gln Pro Gly 145 150 155 160 His Gly Gly Pro Ile Ser ThrAla Asn Tyr Ala Lys Ile Asn Gln Ala 165 170 175 Tyr Gln Ile Ile Gln LysAla Leu Thr Ala Asn Glu Ala Asn Gly Asp 180 185 190 Gly Val Pro Val LeuSer Asp Thr Thr Thr Lys Leu Asp Phe Thr Ile 195 200 205 Gln Gly Asp LysArg Thr Gly Gly Arg Pro Asn Thr Pro Lys Lys Phe 210 215 220 Pro Trp SerAsp Gly Lys Tyr Ile His Thr Gln Trp Ile Asp Thr Thr 225 230 235 240 ProGln Ser Thr Glu Thr Lys Ile Asn Thr Glu Asn Asn Ala Gln Glu 245 250 255Leu Leu Lys Gln Ala Ser Ile Ile Ile Thr Thr Leu Asn Glu Ala Cys 260 265270 Pro Asn Phe Gln Asn Gly Gly Ser Gly Tyr Trp Gln Gly Ile Ser Gly 275280 285 Asn Gly Thr Met Cys Gly Met Phe Lys Asn Glu Ile Ser Ala Ile Gln290 295 300 Gly Met Ile Ala Asn Ala Gln Glu Ala Val Ala Gln Ser Lys IleVal 305 310 315 320 Ser Glu Asn Ala Gln Asn Gln Asn Asn Leu Asp Thr GlyLys Pro Phe 325 330 335 Asn Pro Phe Thr Asp Ala Ser Phe Ala Gln Ser MetLeu Lys Asn Ala 340 345 350 Gln Ala Gln Ala Glu Ile Leu Asn Gln Ala GluGln Val Val Lys Asn 355 360 365 Phe Glu Lys Ile Pro Lys Asn Phe Val SerAsp Ser Leu Gly Val Cys 370 375 380 Tyr Glu Glu Gln Gly Gly Glu Arg ArgGly Thr Asn Pro Gly Gln Val 385 390 395 400 Thr Ser Asn Thr Phe Ala SerGly Cys Ala Tyr Val Glu Gln Thr Ile 405 410 415 Thr Asn Leu Asn Asn SerIle Ala His Phe Gly Thr Gln Glu Gln Gln 420 425 430 Ile Gln Gln Ala GluAsn Ile Ala Asp Thr Leu Val Asn Phe Lys Ser 435 440 445 Arg Tyr Ser GluLeu Gly Asn Thr Tyr Asn Ser Ile Thr Thr Ala Leu 450 455 460 Ser Lys ValPro Asn Ala Gln Ser Leu Gln Asn Val Val Gly Lys Lys 465 470 475 480 AsnAsn Pro Tyr Ser Pro Gln Gly Ile Glu Thr Asn Tyr Tyr Leu Asn 485 490 495Gln Asn Ser Tyr Asn Gln Ile Gln Thr Ile Asn Gln Glu Leu Gly Arg 500 505510 Asn Pro Phe Arg Lys Val Gly Ile Val Ser Ser Gln Thr Asn Asn Gly 515520 525 Ala Met Asn Gly Ile Gly Ile Gln Val Gly Tyr Lys Gln Phe Phe Gly530 535 540 Gln Lys Arg Lys Trp Gly Ala Arg Tyr Tyr Gly Phe Phe Asp TyrAsn 545 550 555 560 His Ala Phe Ile Lys Ser Ser Phe Phe Asn Ser Ala SerAsp Val Trp 565 570 575 Thr Tyr Gly Phe Gly Ala Asp Ala Leu Tyr Asn PheIle Asn Asp Lys 580 585 590 Ala Thr Asn Phe Leu Gly Lys Asn Asn Lys LeuSer Val Gly Leu Phe 595 600 605 Gly Gly Ile Ala Leu Ala Gly Thr Ser TrpLeu Asn Ser Glu Tyr Val 610 615 620 Asn Leu Ala Thr Met Asn Asn Val TyrAsn Ala Lys Met Asn Val Ala 625 630 635 640 Asn Phe Gln Phe Leu Phe AsnMet Gly Val Arg Met Asn Leu Ala Arg 645 650 655 Ser Lys Lys Lys Gly SerAsp His Ala Ala Gln His Gly Ile Glu Leu 660 665 670 Gly Leu Lys Ile ProThr Ile Asn Thr Asn Tyr Tyr Ser Phe Met Gly 675 680 685 Ala Glu Leu LysTyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val 690 695 700 Phe Ala Tyr705 5 20 PRT Helicobacter pylori 5 Glu Asp Asp Gly Phe Tyr Thr Ser ValGly Tyr Gln Ile Gly Glu Ala 1 5 10 15 Ala Gln Met Val 20 6 60 DNAHelicobacter pylori 6 gaagacgacg gcttttacac aagcgtaggc tatcaaatcggtgaagccgc tcaaatggta 60 7 41 PRT Helicobacter pylori 7 Glu Asp Asp GlyPhe Tyr Thr Ser Val Gly Tyr Gln Ile Gly Glu Ala 1 5 10 15 Ala Gln MetVal Thr Asn Thr Lys Gly Ile Gln Asp Leu Ser Asp Asn 20 25 30 Tyr Glu AsnLeu Ser Lys Leu Leu Thr 35 40 8 20 PRT Helicobacter pylori 8 Glu Asp AspGly Phe Tyr Thr Ser Val Gly Tyr Gln Ile Gly Glu Ala 1 5 10 15 Ala GlnMet Val 20

What is claimed is:
 1. An isolated and purified bacterial blood groupanitgen binding adhesin protein (BabA) from Helicobacter pylori species,wherein said protein binds specifically to fucosylated Lewis^(b) type Iand H-1 blood group anitgen-glycoconjugates and, wherein said proteincontains less than 20% bacterial protein impurities, has a molecularweight in the interval of 73 to 75 kDa as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and is not aHopA, HopB, HopC, HopD, or HopE protein.
 2. The adhesion protein ofclaim 1, wherein the SEQ ID NO:5 EDDGFYTSVGYQIGEAAQMV is in an aminoterminal position.
 3. The adhesion protein of claim 1, wherein theprotein has a molecular weight of about 73.5 kDa, as determined bySDS-PAGE.
 4. The adhesion protein of claim 3, wherein said Helicobacterpylori species is Helicobacter pylori strain CCUG
 17875. 5. Animmunogenic composition comprising an adhesion protein according to anyone of claim 1, 2, 3, or 4, optionally together with pharmaceuticallyacceptable excipients.